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Biohydrogenation of unsaturated fatty acids. Presence of dithionite and an endogenous electron donor in Butyrivibrio fibrisolvens.

Two oxygen-consuming substances were isolated from cell-free extracts of the rumen anaerobe, Butyrivibrio fibrisolvens. The major fraction comprising 97% of the total activity was characterized as a three-component mixture of glucose, maltose, and dithionite. The minor activity fraction contained an electron donor for the reduction of cis-9,trans-11-octadecadienoate to trans-11-octadecenoate. After oxidation, the electron donor could be reduced by the dithionite, thereby accounting for the previously observed capacity of cell-free extracts of the bacterium to carry out the biohydrogenation of the conjugated dienoic fatty acid.

Dithionite↗

[Biohydrogenation of erucic acid (22:1 n-9 cis) in an "artificial rumen". II) Effect of pH, potential hydrogen donors and type of anaerobiosis].

The possibility of dietary C18 unsaturated fatty acids double bonds biohydrogenation, which normally occurs in ruminants, has been investigated in the case of erucic acid (22:1 n-9 cis). The results have shown that, while oleic acid is always converted into hydrogenation intermediates and stearic acid, to various extent, erucic acid does not undergo hydrogenation process, unrelated to the incubation conditions applied. Data are discussed on the basis of the different structure of erucic and oleic acids.

Anaerobiosis↗

Biohydrogenation of unsaturated fatty acids. Purification and properties of cis-9,trans-11-octadecadienoate reductase.

The enzyme catalyzing the second step in the biohydrogenation pathway of linoleic acid by Butyrivibrio fibrosolvens cis-9,trans-11-octadecadienoate reductase has been purified to near homogeneity. It has a molecular weight of 60,000 and appears to be a single subunit. The purified enzyme contains 2 mol of iron, 10 mol of fucose, and 12 mol of galactose per 60,000 g. The iron, but not the carbohydrate, is required for enzymatic activity. Phosphatidylethanolamine was also found to be associated with the purified enzyme. Unlike the cell extract that can reduce the double bond of the fatty acid with NADH or alpha-tocopherolquinol as a reductant, the purified enzyme can utilize only alpha-tocopherolquinol. This indicates that another component of the reduction system exists that couples the production of alpha-tocopherolquinol to the oxidation of NADH.

Amino Acids↗

Identification of an endogenous electron donor for biohydrogenation as alpha-tocopherolquinol.

Four fluorescent compounds present in solvent extracts of Butyrivibrio fibrisolvens could serve as electron donors for the biohydrogenation of cis-9,trans-11-octadecadienoate in the presence of dithionite, which was itself inactive. One of the compounds was identified as alpha-tocopherolquinol and another as alpha-tocopherolquinone. A partially purified soluble enzyme preparation from B. fibrisolvens catalyzed the reduction of alpha-tocopherolquinone to alpha-tocopherolquinol in the presence of NADH with a stoichiometry of 1:1. The ratio of alpha-tocopherolquinone produced to fatty acid reduced was 2:1 when the tocopherol derivatives were extracted aerobically. When the extraction was carried out anaerobically, the ratio was 1. It is suggested that the oxidation of 2 molecules of alpha-tocopherolquinol, each to the semiquinone, provides the electrons required for the reduction of the cis-bond of the conjugated dienoic fatty acid. Although alpha-tocopherol, phylloquinol, and reduced menadione are inactive, ubiquinol-4, ubiquinol-10, and trimethylhydroquinone show about one-half the activity of alpha-tocopherolquinol. Plastoquinol and trimethylphytylbenzoquinol are as active as alpha-tocopherolquinol.

Anaerobiosis↗

Identification of deoxy-alpha-tocopherolquinol as another endogenous electron donor for biohydrogenation.

Solvent extracts of Butyrivibrio fibrisolvens contain 2-[3, 7, 11, 15-tetramethylhexadecyl]-3, 5, 6-trimethyl-benzoquinol (deoxy-alpha-tocopherolquinol) that can serve as an alternate electron donor for alpha-tocopherolquinol for the biohydrogenation of cis-9, trans-11-octadecadienoate. In addition, the cell extracts contain deoxy-alpha-tocopherolquinone. This compound arises metabolically from alpha-tocopherolquinone via dehydration to trimethylphytylbenzoquinone followed by hydrogenation to deoxy-alpha-tocopherolquinone. Although the hydrogenation of the isoprene double bond and the conjugated fatty acid both use NADH as the primary reductant, the two reactions appear to be catalyzed by different enzymes.

Electron Transport↗

Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose.

Sixteen batch experiments were performed to evaluate the stability, kinetics, and metabolic paths of heat-shocked digester (HSD) sludge that transforms microcrystalline cellulose into hydrogen. Highly reproducible kinetic and metabolic data confirmed that HSD sludge could stably convert microcrystalline cellulose to hydrogen and volatile fatty acids (VFA) and induce metabolic shift to produce alcohols. We concluded that clostridia predominated the hydrogen-producing bacteria in the HSD sludge. Throughout this study the hydrogen percentage in the headspace of the digesters was greater than 50% and no methanogenesis was observed. The results emphasize that hydrogen significantly inhibited the hydrogen-producing activity of sludge when initial microcrystalline cellulose concentrations exceeded 25.0 g/L. A further 25 batch experiments performed with full factorial design incorporating multivariate analysis suggested that the ability of the sludge to convert cellulose into hydrogen was influenced mainly by the ratio of initial cellulose concentration (So) to initial sludge density (Xo), but not by interaction between the variables. The hydrogen-producing activity depended highly on interaction of So x (So/Xo). Through response surface analysis it was found that a maximum hydrogen yield of 3.2 mmol/g cellulose occurred at So = 40 g/L and So/Xo = 8 g cellulose/g VSS. A high specific rate of 18 mmol/(g VSS-d) occurred at So = 28 g/L and So/Xo = 9 g cellulose/g VSS. These experimental results suggest that high hydrogen generation from cellulose was accompanied by low So/Xo.

Alcohols↗

Thermophilic biohydrogen production from glucose with trickling biofilter.

Thermophilic H2 production from glucose was studied at 55-64 degrees C for 234 days using a continuous trickling biofilter reactor (TBR) packed with a fibrous support matrix. Important parameters investigated included pH, temperature, hydraulic retention time (HRT), and glucose concentration in the feed. The optimal pH and temperature were 5.5 and 60 degrees C, respectively. With decreasing HRT or increasing inlet glucose concentration, volumetric H2 production rate increased but the H2 production yield to glucose decreased gradually. The biogas composition was almost constant at 53 +/- 4% (v/v) of H2 and 47 +/- 4% (v/v) of CO2. No appreciable CH4 was detected when the reactor was under a normal operation. The carbon mass balance showed that, in addition to cell mass, lactate, n-butyrate, CO2, and acetate were major products that comprised more than 85% of the carbon consumed. The maximal volumetric H2 production rate and H2 yield to glucose were 1,050 +/- 63 mmol H2/l.d and 1.11 +/- 0.12 mol H2/mol glucose, respectively. These results indicate that the thermophilic TBR is superior to most suspended or immobilized reactor systems reported thus far. This is the first report on continuous H2 production by a thermophilic TBR system.

Bacteria, Aerobic↗

Influence of substrate concentration on the stability and yield of continuous biohydrogen production.

The effect of substrate concentration (sucrose) on the stability and yield of a continuous fermentative process producing hydrogen was studied. High substrate concentrations are attractive from an energy standpoint as they would minimise the energy required for heating. The reactor was a CSTR; temperature was maintained at 35 degrees C; pH was controlled between 5.2 and 5.3, and the hydraulic retention time (HRT) was 12 h. Online measurements were taken for ORP, pH, temperature, %CO2, gas output and %H2, and data logged using a MatLAB data acquisition toolbox. Steady-state operation was obtained at 10, 20 and 40 g/L of sucrose in the influent, but a subsequent step change to 50 g/L was unsustainable. The hydrogen content ranged between 50% and 60%. The yield of hydrogen decreased as the substrate concentration increased from 1.7 +/- 0.2 mol/mol hexose added at 10 g/L, to 0.8 +/- 0.1 mol/mol at 50 g/L. Sparging with nitrogen improved the hydrogen yield by at least 35% at 40 g/L and at least 33% at 50 g/L sucrose. Sparging also enabled steady-state operation at 50 g/L sucrose. Addition of an extra 4 g/L of n-butyric acid to the reactor operating at 40 g/L sucrose increased the butyrate concentration from 9,830 to 18,900 mg/L, immediately stopping gas production and initiating the production of propionate, whilst the addition of 2 g/L taking the butyrate concentration to 12,200 mg/L did not do so. It was shown that operation at 50 g/L sucrose in a CSTR in butyrate fermentation is possible.

Acetic Acid↗

Biohydrogen production in granular up-flow anaerobic sludge blanket (UASB) reactors with mixed cultures under hyper-thermophilic temperature (70 degrees C).

Hyper-thermophilic hydrogen production without methane was demonstrated for the first time in granular up-flow anaerobic sludge blanket (UASB) system fed with glucose using mixed cultures. The maximum hydrogen yield in this study was 2.47 +/- 0.15 mol H2/mol glucose. This high yield has never been previously reported in mixed culture systems and it was likely due to more favorable thermodynamic conditions at hyper-thermophilic temperatures. Different start-up strategies (bromoethanosulfonate (BES) addition and flow recycle) were evaluated. BES addition during start-up prevented the establishment of methanogenic cultures in granules. Flow recycle was important to achieve higher hydrogen yield through enriching better hydrogen-producing organisms and reduced the start-up period as well. This study indicated UASB system was a promising system for hydrogen production.

Bacteria, Anaerobic↗

Diet and sterol biohydrogenation in the rat: occurrence of epicoprostanol.

The fecal sterols from rats fed several types of semipurified or commercial diets were analyzed by a combination of thin layer and gas liquid chromatography. In rats fed semipurified diets with lard, sucrose, and casein, increasing proportions of lard (0, 8, 20, 65%) enhanced the fecal coprostanol/coprostanol + cholesterol ratio (from 0.50 to 0.85). This ratio was reduced by replacing lard with triolein or a mixture of calcium oleate and linoleate (1:1) and did not change when trierucin was substituted. No coprostanol formation was observed in rats fed a diet with tripalmitin or tristearin. The addition of sodium hyodeoxycholate (0.5%) or cholestyramine (2%) to the basal diet was without effect on the coprostanol/coprostanol + cholesterol ratio in the feces. The addition of sodium taurocholate (0.2, 0.75, and 4%) strongly reduced coprostanol formation, while a chronic bile duct ligation led to an enhancement. Cholesterol feeding (0.05, 0.2, and 0.5% in the diet) slightly increased (from 51 to 66%) coprostanol formation. Trace amounts of epicoprostanol were generally found in the feces. However, in some cases a very high proportion (up to 60%) of this sterol was observed. Possible relationships between the presence of epicoprostanol and the nature of the diet are discussed.

Animals↗

Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae.

When the partial pressure of H(2) was decreased by lowering the total pressure in the headspace of the reactor in a batch fermentation process from 760 mm Hg to 380 mm Hg containing Enterobacter cloacae, the molar yield of H(2) increased from 1.9 mol to 3.9 mol H(2)/mol glucose. The maximum production rate was 0.017 mmol H(2)/h l at 380 mm Hg. The lag period as well as total batch time of H(2) production decreased using a decreased partial pressure.

Air Pressure↗

Biofilm microbial community of a thermophilic trickling biofilter used for continuous biohydrogen production.

Molecular methods were employed to investigate the microbial community of a biofilm obtained from a thermophilic trickling biofilter reactor (TBR) that was operated long-term to produce H(2). Biomass concentration in the TBR gradually decreased as reactor bed height increased. Despite this difference in biomass concentration, samples from the bottom and middle of the TBR bed revealed similar microbial populations as determined by PCR-DGGE analysis of 16S rRNA genes. Nucleotide sequences of most DGGE bands were affiliated with the classes Clostridia and Bacilli in the phylum Firmicutes, and the most dominant bands showed a high sequence similarity to Thermoanaerobacterium thermosaccharolyticum.

Bacillus↗

Biohydrogen production from starch in wastewater under thermophilic condition.

Batch experiments were conducted to convert starch in wastewater into hydrogen at 55 degrees C at various wastewater pH (4.0-9.0) and starch concentrations (9.2-36.6 g/l). The maximum hydrogen yield of 92 ml/g of starch added (17% of the theoretical value) was found at wastewater pH 6.0, and the maximum specific hydrogen production rate of 365 ml/(g-VSS.d) was at wastewater pH 7.0. The methane-free biogas contained up to 60% of hydrogen. The mixed liquor was composed mostly of acetate (40.2-53.4%) and butyrate (26.0-40.9%). Phylogenetic analysis based on 16S rDNA sequences of the 72 clones developed from the sludge at pH 6.0 shows that 85.7% of the clones were closely affiliated with genus Thermoanaerobacterium in family Thermoanaerobacteriaceae; the remaining 14.3% were with an uncultured Saccharococcus sp. clone ETV-T2.

Bacteria↗